This proposal is in response to PA-10-213 titled Development of Assays for High-Throughput screening for use in Probe and Pre-therapeutic discovery. Our overall goal is develop novel biochemical and cell-based assays that will be used to identify inhibitors of Regulator of G protein signaling 17 (RGS17). RGS17 has been identified as being over-expressed in lung and prostate tumors, and facilitates tumor cell growth. Reports indicate that knockdown of RGS17 in lung and prostate cancer cell lines reduces their rate of growth and results in shrinkage of xenograft tumors in nude mice. Unfortunately, there are no reports of small molecule RGS17 inhibitors, and only a handful of reports of small molecules that target any RGS protein. Therefore, RGS17 presents an unexplored target for pre-therapeutic development and represents a novel path to develop lead molecules in the fight against cancer. Thus, the discovery of RGS17 inhibitors would provide a new avenue for both understanding the role of RGS17 in cancer progression as well as provide new small molecule leads for anticancer drug development. In order to facilitate the discovery and optimization of RGS17 inhibitors, we propose to develop assays for an HTS campaign using novel biochemical and cell-based assays. Specifically, we aim to develop the following 1) a high-density (384-1536 well) biochemical assay for the RGS17:G protein alpha subunit protein: protein interaction, and 2) two novel cell based assays for detection of RGS effects on cAMP levels and GIRK channel kinetics in real-time. Achievement of these aims will provide both novel methodology for use in interrogating small molecule libraries for RGS inhibitors in HTS campaigns as well as provide new chemical probes for RGS17 protein function in cancer cell models. After development and validation, our goal is to perform a modest in-house HTS screen to identify and optimize lead molecules as RGS17 inhibitors. The outcome of this project will be both novel screening methodology for RGS protein ligands and new probe molecules that will serve as tools for studying RGS17 protein function and provide initial molecules for pre-therapeutic optimization. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because it aims to probe a novel target, RGS17 using two novel assay methodologies to interrogate small molecule libraries in order to identify probes and pre-therapeutic molecules for studying and treating prostate and lung cancers. RGS17 plays an important role in tumor cell proliferation and has not been targeted in previous campaigns to identify new anticancer leads. Identifying new lead molecules targeting RGS17 is relevant to NIH<s mission of developing new treatments for human diseases.